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Environmental Microbiology Reports (2012)
doi:10.1111/j.1758-2229.2012.00352.x
Occurrence of a specific dual symbiosis in the
excretory organ of geographically distant
Nautiloids populations
emi4_352
1..8
Mathieu Pernice1,2,3* and Renata Boucher-Rodoni2
Coral Reef Ecosystem Laboratory, School of Biological
Sciences, The University of Queensland, Gehrmann
building (#60), Level 7, St Lucia, Queensland 4072,
Australia.
2
UMR 7208 ‘Biologie des ORganismes et Ecosytèmes
Aquatiques’ MNHN-CNRS-IRD-UPMC – Case postale
53, 75231 Paris cedex 05, France.
3
Laboratory for Biological Geochemistry, School of
Architecture, Civil and Environmental Engineering,
Ecole Polytechnique Fédérale de Lausanne, 1015
Lausanne, Switzerland.
Introduction
1
Summary
Nautilus is one of the most intriguing of all sea creatures, sharing morphological similarities with the
extinct forms of coiled cephalopods that evolved
since the Cambrian (542–488 mya). Further, bacterial
symbioses found in their excretory organ are of particular interest as they provide a great opportunity to
investigate the influence of host–microbe interactions upon the origin and evolution of an innovative
nitrogen excretory system. To establish the potential
of Nautilus excretory organ as a new symbiotic
system, it is, however, necessary to assess the specificity of this symbiosis and whether it is consistent
within the different species of present-day Nautiloids.
By addressing the phylogeny and distribution of bacterial symbionts in three Nautilus populations separated by more than 6000 km (N. pompilius from
Philippines and Vanuatu, and N. macromphalus from
New Caledonia), this study confirms the specificity of
this dual symbiosis involving the presence of betaproteobacteria and spirochaete symbionts on a very
wide geographical area. Overall, this work sheds
further light on Nautiloids excretory organ as an innovative system of interaction between bacteria and
cephalopods.
Received 7 December, 2011; revised 16 April, 2012; accepted 23
April, 2012. *For correspondence. E-mail [email protected]; Tel.
(+61) 7336 51964; Fax (+61) 7336 54755.
© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd
Nautilus is one of the most famous of all sea creatures,
inspiring many artists and giving its name to the wondrous
craft imagined by Jules Verne in his novel. However, it is
probably in the eyes of the naturalists that Nautilus has
the most important place, being the last representative of
the subclass of Nautiloidea, and the only extant cephalopod among hundreds of extinct, coiled cephalopod
genera that evolved since the Cambrian (542–488 mya)
(Ward, 1987; Kröger et al., 2011). Despite their morphological similarities with the extinct forms of Ammonoids,
present-day Nautiloids are not ‘living fossils’ but rather
highly specialized animals occupying a specific niche in
the coral reef ecosystems of tropical and subtropical IndoPacific regions. Indeed, Nautiloids live on the external
slope of coral reefs, where they undertake complex vertical migrations (depth ranging from 100 to 700 m) in order
to avoid contact with predators, but also to explore and
scavenge for crustaceans decapods rich in protein
(Ward et al., 1984; Norman, 2000; Dunstan et al., 2011).
After ingestion of their prey, the three steps of digestion
(digestion, absorption and excretion) (Westermann et al.,
2002) lead to the production of ammonia as main endproduct of protein catabolism (Boucaud-Camou and
Boucher-Rodoni, 1983).
Nautiloids excretory system features highly specialized
organs, unique among cephalopods, referred to as pericardial appendages (Fig. 1A and B). These organs are
responsible for most of the excretory processes including
filtration (i.e. filtration of small molecules contained in the
blood), reabsorption (i.e. active reincorporation of compounds from the pericardial coelom to the internal part of
the organ) and secretion of ammonia rich fluid (Schipp
et al., 1985; Mangold et al., 1989). Each pericardial
appendage consists of numerous finger-like villi that
collect the blood from capacious sinuses and produce the
excretory fluid rich in ammonia [up to 200 ppm, Fig. 1B
(Schipp and Martin, 1987)] which is firstly secreted in the
pericardial coelom and then excreted into the mantle
cavity (Mangold et al., 1989). This physiological innovation has a significant impact on Nautilus metabolism and
more particularly on the management of nitrogen waste,
the amount of ammonia released to the surrounding sea
2
M. Pernice and R. Boucher-Rodoni
Fig. 1. Morphology of Nautilus and its
symbiotic organ.
A. Diagram of a longitudinal section through
Nautilus showing the location of the symbiotic
organ referred to as pericardial appendage.
B. Detail of the red inset in A: Simplified
diagram of a pericardial appendage consisting
of numerous finger-like villi. The directions of
blood circulation and secretion of excretory
fluid are indicated by purple and orange
arrows respectively. Modified from Pernice
and colleagues (2007b).
water by Nautilus being three to four times lower than by
any other extant cephalopods (Boucher-Rodoni and
Mangold, 1994).
The bacterial symbioses discovered in Nautilus pericardial appendages about two decades ago (Schipp et al.,
1990) provide a great opportunity to shed light on the
influence of host–microbes interactions on the origin and
evolution of such an innovative excretory system (Fig. 1;
Pernice et al., 2007b). However, before establishing Nautilus pericardial appendages as a new symbiotic system, it
is necessary to assess the specificity of this symbiosis and
whether it is a general rule in present-day Nautiloids.
Currently, information about this symbiosis remains scarce
due to the lack of biological material and the inability to
cultivate the bacterial symbionts artificially. The present
study identifies the specificity of this symbiosis by combin-
ing data concerning the phylogeny and the distribution of
bacterial symbionts in three Nautilus populations separated by more than 6000 km (N. pompilius from Philippines
and Vanuatu, and N. macromphalus from New Caledonia,
Fig. 2). Some aspects concerning the evolution of this
symbiosis and its potential implication in the ecophysiology
of Nautilus excretory organ are further discussed.
Results and discussion
Nautiloid symbiont diversity and phylogeny
Comparison of bacterial 16S rRNA gene sequence analysis of a total of 525 clones indicated the predominance
of two bacterial phylotypes (betaproteobacteria and spirochaetes) in the pericardial appendages of the three
Fig. 2. Collection sites of Nautilus pompilius
(from Philippines and Vanuatu) and
N. macromphalus (from New Caledonia).
© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports
Bacterial symbioses in Nautilus
3
Table 1. Bacterial diversity in the pericardial appendages of N. pompilius (Philippines and Vanuatu) and N. macromphalus (New Caledonia).
Number of partial sequences
S
N
C
Betaproteobacteria
Spirochaeta
Vibrionacae
N. pompilius, Philippines (Pernice et al., 2007a)
Detection using in situ hybridization
3
116
100
28
Yes
36
Yes
52
No
N. pompilius, Vanuatu (this study)
Detection using in situ hybridization
3
114
100
26
Yes
88
Yes
–
No
N. macromphalus, New Caledonia (Pernice et al., 2007b)
Detection using in situ hybridization
3
295
100
177
Yes
118
Yes
–
No
S: number of specimens analysed; N: total number of analysed clones. C: clone library coverage, calculated according to the following equation:
C = 1 - (n/N), where n is the number of unique clones and N is the total number of clones examined (Good, 1953; Ravenschlag et al., 1999). The
number of partial sequences was obtained from cloned PCR product and correspond to 16S rDNA sequence from nucleotide 518 to c.1000 (based
on E. coli numbering). The close association of the different bacterial phylotypes with the host tissue was assessed by in situ hybridization using
specific probes NauBet66 for betaproteobacteria symbionts, NauSpiro255 for spirochaete symbionts (Pernice et al., 2007b) and Gam42a for
Vibrionacae (Manz et al., 1992) as detailed in Text S1. Yes: the bacterial phylotype concerned was detected by in situ hybridization; No: the
bacterial phylotype was not detected.
Nautilus populations analysed. In situ hybridization using
specific probes confirmed that both the betaproteobacteria and the spirochaete symbionts were present and
closely associated to Nautilus tissue in the three different
populations. Another bacterial phylotype belonging to the
gammaproteobacteria (Vibrionacae) was detected in
N. pompilius population from Philippines by amplification
of 16S rRNA gene (Pernice et al., 2007a) but in situ
hybridization analyses using a Gammaproteobacteriaspecific probe [Gam42A (Manz et al., 1992)] failed to
confirm a close association of this bacterial phylotype with
Nautilus tissue. This lack of corroborative evidence calls
into question the symbiotic status of these bacteria
(Table 1). Indeed, the excretory organs of Nautiloids are
connected to the external environment through the pallial
cavity and, therefore, the detection of Vibrionales by polymerase chain reaction (PCR) amplification of 16S rRNA
gene is likely to result from an environmental contamination as previously suggested (Pernice et al., 2007a).
Comparison of the 16SrRNA-gene sequences obtained
for betaproteobacteria and spirochaete symbionts indicates that (i) the sequence variation within each phylotype was remarkably low (below 0.5% based on 1411 bp
of 16S rRNA gene sequence) and (ii) neither the betaproteobacteria nor the spirochaete symbionts were closely
related to any others symbionts associated with other
hosts species or any free-living bacteria (ⱕ 90% of similarity based on 1411 bp of 16S rRNA gene sequence),
supporting the specificity of this dual symbiosis on a very
wide geographical area (Nautilus collection sites being
distant from more than 6000 km, Fig. 2). In addition, phylogenetic analysis of 16S rRNA gene reinforces this
hypothesis by clustering the two phylotypes within two
Nautilus-specific bacterial groups (Fig. 3). Based on the
analysis of 16S rRNA gene, the closest relatives of
the betaproteobacterial symbionts are members of a
clade of free-living ammonia-oxidizing bacteria from the
family Nitrosomonads including Nitrosospria multiformis
and Nitrosospira briensis (Teske et al., 1994). The spirochaete symbionts belong to a monophyletic group that
includes the free-living spirochaetes Spirochaeta bajacaliforniensis (Magot et al., 1997) and Spirochaeta smaragdinae (Fracek and Stolz, 1985) and the symbionts of
gutless marine oligochaetes.
Sequencing and analysis of the gene coding for the 16S
rRNA of Nautilus bacterial symbionts provides new information that may help elucidating the evolutionary history of
Nautiloids and their symbioses. In respect to the evolution
of Nautiloids, the strong genetic similarity observed
between N. macromphalus and N. pompilius bacterial
symbionts supports the hypothesis proposed by Wray and
colleagues (1995) that New Caledonia-endemic species
N. macromphalus, may in fact represent a geographic
variant within a divergent, widespread N. pompilius
species. However, it is now recognized that the gene
coding for the 16S rRNA does not have sufficient resolution
to define the phylogenetic relationships of closely related
bacterial strains (Kowalchuk and Stephen, 2001). Future
co-phylogeny studies should, therefore, use molecular
markers with greater phylogenetic resolution. In this
respect, the gene coding for the glyceraldehyde phosphate
dehydrogenase (gapA) has been recently used to investigate the phylogeography of the mediterranean sepiolids
squid-Vibrio symbioses (Zamborsky and Nishiguchi, 2011)
and could, therefore, represent a promising candidate.
Regarding the origin and evolution of the Nautilusbacteria association, the important genetic distance
between Nautilus symbionts and any bacterial strains referenced in the databases reflects the specificity of this
symbiosis and may suggest a potential host–symbiont
coevolution. Indeed, phylogenetic analyses of 16S rRNA
sequences presented in this study show that all the different Nautilus populations analysed so far share symbiont phylotypes evolutionarily very close (Fig. 3). Further,
© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports
4
M. Pernice and R. Boucher-Rodoni
Fig. 3. The phylogenetic relationships of the spirochaete and betaproteobacterial symbionts of N. pompilius (from Philippines and Vanuatu)
and N. macromphalus (from New Caledonia) inferred from 16S rRNA gene analysis using maximum likelihood (ML) (954 sites analysed).
Numbers at each branch point are the bootstrap values for percentages of 1000 replicate trees calculated by MP (upper) and ML (lower)
methods. Only values > 60% are shown. Trichodesmium thiebautii (cyanobacteria, AF013027) is included as an out-group.
according to the most reliable rates of base substitution in
the 16S rRNA gene for prokaryotes (1% per 50 MA;
Ochman and Wilson, 1987; Moran et al., 1993; Droge
et al., 2006), the large genetic distance (ⱖ 10%) observed
in comparative 16S rRNA gene analysis would suggest (i)
that the last common ancestors of Nautilus symbionts
must have existed earlier than 500 million years ago
(515–630 mya) and (ii) that the diversification of these
symbionts may have begun with their acquisition by the
common ancestor of extant Nautiloids, probably before its
divergence from Coleoids (i.e. squids, cuttlefish and octopods) at the Silurian/Devonian boundary (416 ⫾ 60 mya)
(Kröger et al., 2011). However, the rates of molecular
evolution can vary considerably among bacterial lineages
(Ochman et al., 1999) and the use of such a fixed rate of
base substitution is likely to result in biased estimates
(Kuo and Ochman, 2009). It is reasonable to suggest that
this association occurred long ago, but in order to better
understand when this symbiosis evolved, further work
should focus on different species of Nautiloids that have
never been analysed concerning their potential symbiotic
populations, including N. belauensis, N. stenomphalus,
and N. repertus, but also and most importantly, Allonautilus scrobitulatus which is the only species of the genus
Allonautilus (Wray et al., 1995; Ward and Saunders,
1997). In this respect, the study of Nautiloids populations
living off New Guinea, the only place in the world where
N. pompilius is sympatric with A. scrobiculatus, would be
of the greatest interest as it could allow comparing bacterial symbionts in two divergent lineages of Nautiloids
living within the same environment.
Distribution of bacterial symbionts in relation to the
ecophysiology of the symbiotic organ
Imaging of the bacterial symbionts in Nautilus pericardial
appendages by using in situ hybridization (Text S1)
revealed a remarkably stable spatial distribution in the
different Nautilus bacterial populations, with two levels of
structuration within the host tissue (Fig. 4). A first level of
© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports
Bacterial symbioses in Nautilus
5
Fig. 4. Distribution of bacterial symbionts in
N. pompilius from Philippines (A, D, G) and
Vanuatu (B, E, H) and N. macromphalus from
New Caledonia (C, F, I).
A–C. Longitudinal section through a villus and
general distribution of the symbiotic bacteria
(CARD-FISH with eubacterial probe EUB388
in green) in N. pompilius from Philippines (A)
and Vanuatu (B) and N. macromphalus from
New Caledonia (C).
D–I. Transversal sections of a pericardial
villus and multicolour CARDFISH images of
bacterial symbionts in N. pompilius from
Philippines (D, G) and Vanuatu (E, H) and
N. macromphalus from New Caledonia (F, I).
The spirochaete symbionts (NauSpiro255
probe in red) are closely associated with the
pericardial epithelium in peripheral areas. The
betaproteobacterial symbionts (NauBet66
probe in green) are found in peripheral and
invaginated areas, and are less closely
associated with the epithelium. In blue,
Nautilus tissue stained with DAPI.
Scale bars: A–C = 500 mm; D–F = 50 mm,
G–I = 10 mm.
organization was observed by using a probe recognizing
most bacteria [Eub388 (Amann et al., 1990)] in longitudinal section of the excretory organs and concerned the
predominance of bacterial symbionts in the baso-median
region of the villi and their complete absence in the apical
region (Fig. 4A–C). A second level of spatial distribution,
specific to each group of symbionts, was revealed by
using the spirochaete symbiont-specific probe NauSpiro255 and the Betaproteobacterium-specific probe
NauBet66 (Pernice et al., 2007b) and indicated the predominance of betaproteobacteria symbionts in the cavities formed by baso-medial invaginations of the villi while
the spirochaete symbionts were mainly present in the
peripheral areas (Fig. 4D–I). In accordance with the functional organization revealed by the ultrastructure of the
pericardial villis (Schipp et al., 1985), this spatial distribution suggests that the bacterial population may interact
specifically with the host tissue for two main reasons.
First, the bacterial symbionts are concentrated and
attached to the outer epithelium in the baso-medial region
of the pericardial villi which is highly active in the ultrafiltration and the reabsorption processes while the apical
part of the villi is devoid of any symbionts (Schipp and
Martin, 1987). Second, the ultrastructure of the outer epithelium and its polar organization in the symbiotic region
of pericardial villi are characteristic of an energy-requiring,
transport active tissue with a high density of mitochondria
and ionic pumps (Martin, 1983; Pernice et al., 2007c). The
precise distribution of the bacterial symbionts in this transport active region of the excretory tissue is likely to reflect
metabolic interactions with their host tissue based on a
three-step process: (i) the secretion of the excretory fluid
by the host tissue; (ii) the degradation and assimilation of
compounds present in the excretory fluid by the bacterial
symbionts; and (iii) the reabsorption of compounds
derived from bacterial assimilation by the host tissue
(Fig. 5). The ecophysiology of this symbiotic system
seems to be primarily governed by pH and ammonia
© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports
6
M. Pernice and R. Boucher-Rodoni
Fig. 5. Ecophysiological model of Nautilus symbiotic organ.
The arrows indicate the main metabolic processes driving the
ecophysiology of the symbiotic system: filtration of molecules
circulating in the blood (purple arrow); secretion of the excretory
fluid acid and rich in ammonia (orange arrow); assimilation of
compounds present in the excretory fluid by the bacterial symbionts
(blue arrow); and reabsorption of compounds derived from bacterial
assimilation by the host tissue (black arrow). The rod shapes in
green (betaproteobacteria) and the red dots (spirochaetes) illustrate
the location of symbiotic bacteria. The large arrow in orange
indicates the gradient of ammonia concentration and pH due to the
secretion of excretory fluid.
concentration but other factors could be involved, such as
the low concentrations of oxygen present in the cavities of
villis (Schipp et al., 1990) or the high levels of heavy
metals in pericardial appendages tissue (Bustamante
et al., 2000; Pernice et al., 2009). These factors could
ultimately contribute to the specific distribution of each
group of symbionts, the betaproteobacteria and spirochaete symbionts establishing their own ecological niche
within this micro-ecosystem.
Bacterial symbioses involved in the recycling of digestive or waste products have been described in a number
of metazoans during the past decades and their activities
are now widely recognized as essential for the functioning
of all ecosystems including the human body (Turnbaugh
et al., 2006; Douglas, 2009; Wagner, 2009). In Nautilus,
the identity of the compounds involved in the host–
symbiont interaction remains unclear. Most of the preliminary work has been focused on the potential role of
bacterial symbionts in nitrogen metabolism as (i) Nautilus
betaproteobacteria symbionts are phylogenetically affiliated to Nitrosomonadaceae, an ammonia-oxidizing
lineage; (ii) the symbiotic bacteria live in an ammonia-rich
environment, ammonia being the main end-product of
Nautilus excretion (Martin, 1983); and (iii) transformation
of ammonia could have an important ecological role for
Nautilus by detoxifying its tissue and/or by providing the
nitrogen gas filling its chambered shell (containing over
90% of nitrogen as gas), responsible for its neutral buoyancy (Denton, 1974; Boucher-Rodoni and Mangold,
1994).
Preliminary results concerning the potential implication
of bacterial symbionts in the transformation of ammonia
are contrasted. Indeed, a molecular approach using PCR
amplification has failed to detect the presence of genes
coding for enzymes related to nitrogen metabolism such
as amoA (Purkhold et al., 2000), nirK (Braker et al., 1998)
and nosZ (Scala and Kerkhof, 1998) in the DNA extracted
from Nautilus pericardial appendages (Pernice et al.,
2007b). A second approach, using isotopic incubation of
the symbiotic organ in seawater enriched in 15Nammonia and 14N-nitrate (Text S1) has highlighted a
series of interesting metabolic responses including rapid
production of nitrites (in less than 6 h) followed by nitrites
assimilation (from 12 to 18 h) and small but significant
accumulation of 15N-labelled nitrogenous gas (Fig. S1).
Such metabolic responses could indicate a combination
of nitrification (i.e. oxidation of ammonia to nitrite) and
further denitrification (oxidation of nitrite to dinitrogen gas)
suggesting that the two bacterial symbionts may share a
mutualistic relationship with each other in an endosymbiotic nitrogen cycle, in addition to their symbiotic relationship with Nautilus host. However, the rate of 15N-labelled
nitrogenous gas production observed (c. 0.3 nM/h) was
remarkably low in comparison to the rate of nitrite assimilation (c. 8 mM/h), which calls into question this hypothesis
of nitrification combined to denitrification. An alternative
hypothesis concerning the metabolic pathways of the
symbionts is that the symbiotic bacteria could allow Nautilus to better conserve nitrogen by degrading proteins
present in the coelomic fluid into amino acids. Such metabolic activity could ultimately facilitate the reabsorption
process in baso-median region of Nautilus pericardial
appendages (Pernice et al., 2007b). Further investigations are clearly needed to understand how the betaproteobacteria and spirochaete symbionts are involved in
Nautilus excretion but, given their high density in the host
tissue (approx. 150 ¥ 106 cells per gram of fresh tissue;
Pernice et al., 2007c), it is likely that these symbiotic
populations may have a profound effect not only on the
function but also on the development of the excretory
organs of present-day Nautiloids.
Acknowledgements
We thank the Aquarium and the centre IRD of Noumea (New
Caledonia) as well as the Maritime college of Luganville
© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports
Bacterial symbioses in Nautilus
(Vanuatu) and Aquascapes (Philippines) for their help in
providing N. macromphalus and N. pompilius specimens,
respectively. Dr N. Dubilier and Dr G. Lavik are gratefully
acknowledged for their help in molecular and isotopic analyses respectively. We would like to thank Dr O. Pantos and Dr
S. Dunn for their helpful comments and edits on the manuscript and Dr P. Joannot for her continuous support. MP was
supported by the French Ministry for National Education and
Research and the Max Planck Institute for Marine Microbiology during his PhD and by a Marie Curie International Outgoing Fellowship during his postdoctoral research. Additional
funding was provided by the University Pierre-et-Marie Curie,
the CNRS and a research Grant from the Government of New
Caledonia awarded by the Pacific Fund.
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Supporting information
Additional Supporting Information may be found in the online
version of this article:
Fig. S1. Metabolic activity of Nautilus bacterial symbionts in
seawater enriched in labelled nitrogen compounds
(ammonia, 15NH4+; nitrate, 14NO3-).
Text S1. Supplementary information for Material and
Methods.
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